目的： 目前有越來越多的研究發現，S100家族中的DNA甲基化程度會進一步影響腫瘤的增殖侵襲以及轉移。本研究主旨在探討S100A15以及其啟動子DNA甲基化模式在肺癌發展中的功能作用。
實驗設計： 我們分析了來自178個肺癌病人中的115個肺腺癌患者的福爾馬林固定石蠟包埋標本，其中包括24例早期腺癌和91例晚期肺腺癌病人組織切片標本。使用較不具轉移性（CL1-0）和高轉移性（CL1-5）肺腺癌細胞株進行體外實驗。經由免疫組織化學染色分析，來測定S100A15蛋白的表現量，並通過焦磷酸測序法測定其DNA甲基化程度。
結果： 針對組織切片檢體進行核染色，其中發現具有遠處轉移的肺腺癌病人這組和不具遠處轉移的肺腺癌及晚期與早期患者比較，這兩組相較中，遠處轉移性及晚期患者肺腺癌患者S100A15核染色顯著增加。 在使用第一線標靶藥物之肺腺癌患者中標記較高s100a15核表現的第1/3年的整體存活率降低。
在甲基化程度檢測方面，在轉移性肺腺癌患者中，S100A15啟動子區-423 / -248/-412 CpG位點DNA甲基化呈現下降，並且一年存活率也較低。
在細胞株方面， 相對CL1-0, CL1-5細胞株在-412 / -248 / -56 CpG位點顯示S100A15啟動子DNA甲基化降低，S100A15基因/蛋白表現量增加。
將CL1-5細胞株中S100A15基因刪除，則抑制癌細胞增殖，遷移和侵襲，而S100A15過表現的CL1-0細胞株中則促進癌細胞增殖，遷移和侵襲。
RNA次世代定序分析顯示S100A15的上調會影響CTNNB1，ZEB1，CDC42，HSP90AA1，BST2和PCNA的關鍵調控。
結論： S100A15啟動子區域的過表現和DNA低甲基化可能經由以CTNNB1 為中心的途徑引起肺腺癌腫瘤高轉移能力。

Purpose: Human S100A15 is up-regulated in several human cancers. We investigate the relations between S100A15 and its promoter DNA methylation patterns in lung adenocarcinoma progression.
Material and Methods: We analyzed 115 patients with lung adenocarcinoma (24 early stage and 91 advanced stage) in total 178 formalin-fixed paraffin embedded specimens from lung cancer patients. The less invasive (CL1-0) and highly invasive (CL1-5) lung adenocarcinoma (AC) cell lines were used for in vitro experiments. Then, S100A15 protein expression is evaluated by immunohistochemistry stain. S100A15 promoter DNA methylation levels were measured by pyrosequencing.
Results: S100A15 nuclear staining was increased in lung AC patients with distant metastasis versus those without distant metastasis and with advanced stage versus early stage. There was reduced one/ three-year overall survival in the AC patients receiving fist line target therapy and harboring high nuclear expressions of S100A15 versus harboring low nuclear expressions of S100A15. S100A15 promoter DNA methylation levels over -423/-412/-248 CpG sites were decreased in AC patients with distant metastasis versus those without distant metastasis, and the former associated with lower one-year overall survival. CL1-5 cell lines display decreased S100A15 promoter DNA methylation over –412/-248 /-56 CpG sites and increased S100A15 gene/protein expressions versus CL1-0 cell lines. Knockdown of S100A15 in CL1-5 cell line inhibited cancer proliferation, migration, and invasion, while over-expression of S100A15 in CL1-0 cell line promoted cancer proliferation, migration, and invasion. RNA sequencing analysis revealed potential biological effects of S100A15 with CTNNB1, ZEB1, CDC42, HSP90AA1, BST2, and PCNA being the pivotal down-stream mediators.
Conclusions: S100A15 over-expression and DNA hypomethylation of its promoter region were associated with high metastasis potential and poor outcome in lung AC, probably through triggering CTNNB1-centered pathways.

目 錄
論文審定書 ………………………………………………………… i
中文摘要 …………………………………………………………… ii
英文摘要 ………………………………………………….………… iv
第 一 章 前言…………………………………………………… 1
第 二 章 材料與方法…………………………………………… 9
第 三 章 結果…………………………………………………… 25
第 四 章 討論 …………………………………………………29
第 五 章 圖表 …………………………………………………33

參考文獻……………………………………………………………46
附錄 … ……………………………………………………………… 50

[1] Ministry of Health and Welfare, Taiwan, ROC. Cancer Registry
Annual Report. 2014.
[2] Yin J, Fu W, Dai L, Jiang Z, Liao H, Chen W, et al. ANKRD22 promotes progression of non-small cell lung cancer through transcriptional up-regulation of E2F1. Scientific reports. 2017;7:4430.
[3] Wood SL, Pernemalm M, Crosbie PA, Whetton AD. The role of the tumor-microenvironment in lung cancer-metastasis and its relationship to potential therapeutic targets. Cancer treatment reviews. 2014;40:558-66.
[4] Ramalingam SS, Owonikoko TK, Khuri FR. Lung cancer: New biological insights and recent therapeutic advances. CA: a cancer journal for clinicians. 2011;61:91-112.
[5] De Wever W. Role of integrated PET/CT in the staging of non-small cell lung cancer. JBR-BTR : organe de la Societe royale belge de radiologie. 2009;92:124-6.
[6] Navani N, Nankivell M, Lawrence DR, Lock S, Makker H, Baldwin DR, et al. Lung cancer diagnosis and staging with endobronchial ultrasound-guided transbronchial needle aspiration compared with conventional approaches: an open-label, pragmatic, randomised controlled trial. The Lancet Respiratory medicine. 2015;3:282-9.
[7] Silvestri GA, Gonzalez AV, Jantz MA, Margolis ML, Gould MK, Tanoue LT, et al. Methods for staging non-small cell lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest. 2013;143:e211S-e50S.
[8] Le HT, Nguyen HT, Min HY, Hyun SY, Kwon S, Lee Y, et al. Panaxynol, a natural Hsp90 inhibitor, effectively targets both lung cancer stem and non-stem cells. Cancer letters. 2018;412:297-307.
[9] Li H, Ren Y, Xia L, Qu R, Kong L, Yin Z, et al. Association of MicroRNA-149 Polymorphism with Lung Cancer Risk in Chinese Non-Smoking Female: A Case-Control Study. PloS one. 2016;11:e0163626.
[10] Goldstraw P, Chansky K, Crowley J, Rami-Porta R, Asamura H, Eberhardt WE, et al. The IASLC Lung Cancer Staging Project: Proposals for Revision of the TNM Stage Groupings in the Forthcoming (Eighth) Edition of the TNM Classification for Lung Cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2016;11:39-51.
[11] Chan BA, Hughes BG. Targeted therapy for non-small cell lung cancer: current standards and the promise of the future. Translational lung cancer research. 2015;4:36-54.
[12] Chen YC, Hsiao CC, Chen KD, Hung YC, Wu CY, Lie CH, et al. Peripheral immune cell gene expression changes in advanced non-small cell lung cancer patients treated with first line combination chemotherapy. PloS one. 2013;8:e57053.
[13] Tian T, Li X, Hua Z, Ma J, Liu Z, Chen H, et al. S100A1 promotes cell proliferation and migration and is associated with lymph node metastasis in ovarian cancer. Discovery medicine. 2017;23:235-45.
[14] Lesniak W, Graczyk-Jarzynka A. The S100 proteins in epidermis: Topology and function. Biochimica et biophysica acta. 2015;1850:2563-72.
[15] Lindsey JC, Lusher ME, Anderton JA, Gilbertson RJ, Ellison DW, Clifford SC. Epigenetic deregulation of multiple S100 gene family members by differential hypomethylation and hypermethylation events in medulloblastoma. British journal of cancer. 2007;97:267-74.
[16] Lesniak W. Epigenetic regulation of S100 protein expression. Clinical epigenetics. 2011;2:77-83.
[17] Lee J, Wysocki PT, Topaloglu O, Maldonado L, Brait M, Begum S, et al. Epigenetic silencing of S100A2 in bladder and head and neck cancers. Oncoscience. 2015;2:410-8.
[18] Wolf R, Voscopoulos C, Winston J, Dharamsi A, Goldsmith P, Gunsior M, et al. Highly homologous hS100A15 and hS100A7 proteins are distinctly expressed in normal breast tissue and breast cancer. Cancer letters. 2009;277:101-7.
[19] Liu J, Li X, Dong GL, Zhang HW, Chen DL, Du JJ, et al. In silico analysis and verification of S100 gene expression in gastric cancer. BMC cancer. 2008;8:261.
[20] Hou S, Tian T, Qi D, Sun K, Yuan Q, Wang Z, et al. S100A4 promotes lung tumor development through beta-catenin pathway-mediated autophagy inhibition. Cell death & disease. 2018;9:277.
[21] Li A, Gu Y, Li X, Sun H, Zha H, Xie J, et al. S100A6 promotes the proliferation and migration of cervical cancer cells via the PI3K/Akt signaling pathway. Oncology letters. 2018;15:5685-93.
[22] Wang XH, Du H, Li L, Shao DF, Zhong XY, Hu Y, et al. Increased expression of S100A6 promotes cell proliferation in gastric cancer cells. Oncology letters. 2017;13:222-30.
[23] Tian T, Li X, Hua Z, Ma J, Wu X, Liu Z, et al. S100A7 promotes the migration, invasion and metastasis of human cervical cancer cells through epithelial-mesenchymal transition. Oncotarget. 2017;8:24964-77.
[24] Liu C, Xing G, Wu C, Zhu J, Wei M, Liu D, et al. Inhibition of Expression of the S100A8 Gene Encoding the S100 Calcium-Binding Protein A8 Promotes Apoptosis by Suppressing the Phosphorylation of Protein Kinase B (Akt) in Endometrial Carcinoma and HEC-1A Cells. Medical science monitor : international medical journal of experimental and clinical research. 2018;24:1836-46.
[25] Crutcher RJ. CAPAS 2.0: a computer tool for coding transcribed and digitally recorded verbal reports. Behavior research methods. 2007;39:167-74.
[26] Katono K, Sato Y, Jiang SX, Kobayashi M, Saito K, Nagashio R, et al. Clinicopathological Significance of S100A10 Expression in Lung Adenocarcinomas. Asian Pacific journal of cancer prevention : APJCP. 2016;17:289-94.
[27] Suzuki S, Yamayoshi Y, Nishimuta A, Tanigawara Y. S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells. Proteome science. 2011;9:76.
[28] Woo T, Okudela K, Mitsui H, Tajiri M, Rino Y, Ohashi K, et al. Up-Regulation of S100A11 in Lung Adenocarcinoma - Its Potential Relationship with Cancer Progression. PloS one. 2015;10:e0142642.
[29] Funk S, Mark R, Bayo P, Flechtenmacher C, Grabe N, Angel P, et al. High S100A8 and S100A12 protein expression is a favorable prognostic factor for survival of oropharyngeal squamous cell carcinoma. International journal of cancer. 2015;136:2037-46.
[30] Li D, Zeng Z, Yu T, Qin J, Wu J, Song JC, et al. Expression and clinical implication of S100A12 in gastric carcinoma. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2016;37:6551-9.
[31] Zhong J, Liu C, Chen YJ, Zhang QH, Yang J, Kang X, et al. The association between S100A13 and HMGA1 in the modulation of thyroid cancer proliferation and invasion. Journal of translational medicine. 2016;14:80.
[32] Qian J, Ding F, Luo A, Liu Z, Cui Z. Overexpression of S100A14 in human serous ovarian carcinoma. Oncology letters. 2016;11:1113-9.
[33] Zhu M, Wang H, Cui J, Li W, An G, Pan Y, et al. Calcium-binding protein S100A14 induces differentiation and suppresses metastasis in gastric cancer. Cell death & disease. 2017;8:e2938.
[34] Buchau AS, Hassan M, Kukova G, Lewerenz V, Kellermann S, Wurthner JU, et al. S100A15, an antimicrobial protein of the skin: regulation by E. coli through Toll-like receptor 4. The Journal of investigative dermatology. 2007;127:2596-604.
[35] Liu J, Guo Y, Fu S, Yang M, Sun KL, Fu WN. Hypomethylation-induced expression of S100A4 increases the invasiveness of laryngeal squamous cell carcinoma. Oncology reports. 2010;23:1101-7.
[36] Hattinger E, Zwicker S, Ruzicka T, Yuspa SH, Wolf R. Opposing functions of psoriasin (S100A7) and koebnerisin (S100A15) in epithelial carcinogenesis. Current opinion in pharmacology. 2013;13:588-94.
[37] Chu YW, Yang PC, Yang SC, Shyu YC, Hendrix MJ, Wu R, et al. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American journal of respiratory cell and molecular biology. 1997;17:353-60.
[38] Sarkar S, Horn G, Moulton K, Oza A, Byler S, Kokolus S, et al. Cancer development, progression, and therapy: an epigenetic overview. International journal of molecular sciences. 2013;14:21087-113.
[39] Hsu YL, Hung JY, Liang YY, Lin YS, Tsai MJ, Chou SH, et al. S100P interacts with integrin alpha7 and increases cancer cell migration and invasion in lung cancer. Oncotarget. 2015;6:29585-98.
[40] Rehman I, Cross SS, Azzouzi AR, Catto JW, Deloulme JC, Larre S, et al. S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors. British journal of cancer. 2004;91:739-44.
[41] Yao R, Lopez-Beltran A, Maclennan GT, Montironi R, Eble JN, Cheng L. Expression of S100 protein family members in the pathogenesis of bladder tumors. Anticancer research. 2007;27:3051-8.
[42] Medina-Franco JL, Mendez-Lucio O, Duenas-Gonzalez A, Yoo J. Discovery and development of DNA methyltransferase inhibitors using in silico approaches. Drug discovery today. 2015;20:569-77.
[43] Tanaka H, Ogishima S. Network biology approach to epithelial-mesenchymal transition in cancer metastasis: three stage theory. Journal of molecular cell biology. 2015;7:253-66.
[44] Wang L, Kong W, Liu B, Zhang X. Proliferating cell nuclear antigen promotes cell proliferation and tumorigenesis by up-regulating STAT3 in non-small cell lung cancer. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2018;104:595-602.
[45] Wang CI, Chen YY, Wang CL, Yu JS, Chang YS, Yu CJ. mTOR regulates proteasomal degradation and Dp1/E2F1- mediated transcription of KPNA2 in lung cancer cells. Oncotarget. 2016;7:25432-42.
[46] Zheng C, Wang Y, Yang L, Zhou S, Gao Y, Li F, et al. Cell Division Cycle 42 plays a Cell type-Specific role in Lung Tumorigenesis. Scientific reports. 2017;7:10407.
[47] Wainwright DA, Balyasnikova IV, Han Y, Lesniak MS. The expression of BST2 in human and experimental mouse brain tumors. Experimental and molecular pathology. 2011;91:440-6.